Xiaoran Roger Liu1, Mengru Mira Zhang1, Don L Rempel1, Michael L Gross2. 1. Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, MO, 63130, USA. 2. Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, MO, 63130, USA. mgross@wustl.edu.
Abstract
We report a novel method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) to characterize protein-ligand binding stoichiometry, binding sites, and site-specific binding constants. The system used to test the method is melittin-calmodulin, in which the peptide melittin binds to calcium-bound calmodulin. Global-level measurements reveal the binding stoichiometry of 1:1 whereas peptide-level data coupled with fitting reveal the binding sites and the site-specific binding affinity. Moreover, we extended the analysis to the residue level and identified six critical binding residues. The results show that melittin binds to the N-terminal, central linker, and C-terminal regions of holo-calmodulin with an affinity of 4.6 nM, in agreement with results of previous studies. LITPOMS, for the first time, brings high residue-level resolution to affinity measurements, providing simultaneously qualitative and quantitative understanding of protein-ligand binding. The approach can be expanded to other binding systems without tagging the protein to give high spatial resolution. Graphical Abstract.
We report a novel method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) to characterize protein-ligand binding stoichiometry, binding sites, and site-specific binding constants. The system used to test the method is melittin-n class="Gene">calmodulin, in which the peptide melittin binds to calcium-bound calmodulin. Global-level measurements reveal the binding stoichiometry of 1:1 whereas peptide-level data coupled with fitting reveal the binding sites and the site-specific binding affinity. Moreover, we extended the analysis to the residue level and identified six critical binding residues. The results show that melittin binds to the N-terminal, central linker, and C-terminal regions of holo-calmodulin with an affinity of 4.6 nM, in agreement with results of previous studies. LITPOMS, for the first time, brings high residue-level resolution to affinity measurements, providing simultaneously qualitative and quantitative understanding of protein-ligand binding. The approach can be expanded to other binding systems without tagging the protein to give high spatial resolution. Graphical Abstract.
Entities:
Keywords:
Binding affinity; Fast photochemical oxidation of proteins (FPOP); LITPOMS; Ligand titration; Melittin Calmodulin; Site-specific binding
Authors: Que N Van; Cesar A López; Marco Tonelli; Troy Taylor; Ben Niu; Christopher B Stanley; Debsindhu Bhowmik; Timothy H Tran; Peter H Frank; Simon Messing; Patrick Alexander; Daniel Scott; Xiaoying Ye; Matt Drew; Oleg Chertov; Mathias Lösche; Arvind Ramanathan; Michael L Gross; Nicolas W Hengartner; William M Westler; John L Markley; Dhirendra K Simanshu; Dwight V Nissley; William K Gillette; Dominic Esposito; Frank McCormick; S Gnanakaran; Frank Heinrich; Andrew G Stephen Journal: Proc Natl Acad Sci U S A Date: 2020-09-10 Impact factor: 11.205